Non-competitive immunoassay with blocking of unoccupied specific binding sites on solid phase

ABSTRACT

A non-competitive method for the determination of analytes. Initially the analyte is bound to a specific binding partner, after which the unoccupied binding sites of the binding partner are inactivated. The bound analyte is then dissociated from the binding partner and replaced by a labeled marker, after which the bound labeled marker is determined. The signal from the bound labeled marker is directly proportional to the initial amount of analyte in the sample, which makes the present method more favorable than the competitive assays.

This application is a U.S. national stage application of InternationalApplication PCT/FI97/00059, filed Feb. 4, 1997, and claims benefit ofthe Feb. 6, 1996, filing date of Finnish patent application No. 960,534.

This invention relates to a method for the non-competitive determinationof analytes.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details with respect to the practice, are incorporated byreference.

The application of biospecific binding partners for the determination ofanalytes from complex samples has gained widespread use in in vitrodiagnostics. At present, most of such determinations useantibodies--either polyclonal or monoclonal--as the biospecific bindingpartner. The determinations that use antibodies are often calledimmunoassays. Immunoassays are often divided into non-competitive andcompetitive ones, where non-competitive assays involve the use of anexcess of reagents and two biospecific binding partners binding to thesame analyte (this type of assay is commonly called the sandwich assay).Competitive assays on the other hand rely on the measurement of theratio between the free and bound labelled marker, with the ratio beingmodified by the amount of the analyte in the sample.

In usual non-competitive immunoassays, a sample containing the antigento be determined is incubated with an excess of a capture antibodyimmobilized to a solid support. A labelled antibody, specific foranother epitope on the same antigen, is added in excess. A sandwichcomprising "catching antibody--antigen--labelled antibody" is thusformed. After completion of the incubation, the unbound labelledantibody is removed and the signal from the label in the sandwich ismeasured. The signal is thus directly proportional to the antigenconcentration in the sample.

The above non-competitive method cannot, however, be easily applied tothe determination of small molecular weight analytes. The smallmolecular weight analyte is too small to simultaneously bind to twodifferent antibodies. The immunoassay of these analytes is thereforenormally performed by a competitive assay. In a typical implementationof competitive assay, the sample containing the analyte to be determinedas well as a labelled derivative of the analyte are added to animmobilized antibody specific for said analyte. The unlabelled andlabelled analytes compete for the binding sites on the antibody. Aftercompletion of the incubation, unbound analytes are removed and thesignal from the bound labelled analyte is detected. Increasedconcentrations of the analyte in the sample will thus result in adecreased signal. When the signal strength is plotted as a function ofincreasing analyte concentration, a sigmoidal, descending curve isobtained. The sensitivity of this competitive assay is not as high asthat of non-competitive assays due to the fact that the signal is at itshighest value at the zero dose. As Ekins et al. [1,2] have pointed out,sensitivity can be defined as the smallest dose distinguishable from thezero dose. A usual criterion for this distinction is that the signal ofthe dose differs by more that two standard deviations (SD) from thesignal of the zero dose. In a competitive assay one has to detect asmall difference between two high signals, whereas in a non-competitiveassay one has to detect a small difference between two low signals. Asthe SD of the low signal tends to be less than that of the high signal,the non-competitive assay is able to detect smaller differences insignal than the competitive assay. Assuming that the slopes of thedose-response curves of both assays have the same absolute value in thelow dose range, then non-competitive assay is more sensitive, becauseits ability to detect smaller differences in signal is in directproportion to its ability to detect smaller differences in the dose,too.

We have recently discovered a new non-competitive method for thedetermination of small molecular weight analytes such as haptens.Contrary to the usually employed competitive assay described above, thenew method gives a linear, ascending curve when signal strength isplotted as a function of increasing analyte concentration. This keyfeature of the new method makes it more sensitive than the competitiveimmunoassay.

SUMMARY OF THE INVENTION

This invention relates to a non-competitive method for the determinationof analytes, comprising the steps of

a) contacting a sample containing the analyte with a binding partnerspecific for said analyte,

b) adding a blocker, said blocker being able to inactivate the bindingsites of said binding partner that are unoccupied, but not being able toinactivate the binding sites of said binding partner that are occupiedby the analyte,

c) dissociating the bound analyte from the binding partner,

d) adding a labelled marker which is able to occupy the binding sitesfrom which the analyte was dissociated, but which is not able to occupythe binding sites that were inactivated by the blocker, wherein step dcan be performed subsequently to or simultaneously with step c, and

e) measuring the signal from the labelled marker bound to the bindingpartner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the signal strength versus concentration of 17β-Estradiolin the range 0 to 0.5 nM measured according to the method of thisinvention.

FIG. 1B shows the signal strength versus concentration of 17β-Estradiolin the range 0 to 2 nM measured according to the method of thisinvention.

In both figures the filled squares represent replacement time of 10minutes and the open squares represent replacement time of 5 minutes.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this invention the term "analyte" shall mean anymolecule for which there exists a specific binding partner. The presentinvention is especially suited for the determination small (i.e.molecular weight less than 5000 Daltons) analytes. Such analytes arecommon among the groups like steroids, vitamins, prostaglandins,antiasthmatic drugs, antiarrythmic drugs, antineoplastic drugs,anticonvulsant drugs, antibiotics, antiarthritic drugs, antidepressantdrugs, and drugs of abuse such as cocaine, morphine, heroin,amphetamine, methamphetamine, cannabinoids and the like, andenvironmental pollutants and toxins.

The "binding partner" is defined as an entity which has sufficientaffinity and specificity for the analyte. Typical binding partners aremacromolecules such as proteins and nucleic acids.

Proteins which are particularly suitable as binding partners includeantibodies and receptors. Antibodies may be raised in animals [3] orthey may be selected from recombinant libraries [4]. Antibodies specificfor important analytes are commercially available from various sources.Receptors are naturally present in humans and other organisms, and theymay be isolated from these sources. Alternatively, the genes coding forthe receptors may be cloned by recombinant DNA technology [5],transferred to appropriate host organisms and expressed there to producethe receptor of interest [5,6]. Other naturally occurring proteins whichmay be suitable as binding partners are various carrier and transportingproteins, e.g. sex hormone binding globulin (SHBG). If no suitablebinding partner is found among naturally existing proteins, one may becreated by protein engineering, either de novo or using an existingprotein as a starting material. Such protein engineering would involveeither genetic engineering or chemical modifications to the protein orboth.

The term "blocker" shall mean any substance that prevents the unoccupiedbinding partner from binding the labelled marker, but which does notprevent the occupied binding partner from liberating the analyte boundthereto and subsequently binding the labelled marker.

Preferably the blocker is a molecule exhibiting the same or similarepitope as the analyte to be determined, resulting in a mutuallyexclusive binding of the analyte and the blocker to the binding partner.The blocker is thus preferably, but not necessarily, a derivative of theanalyte.

The blocker may also be an antibody that binds to the binding partneronly when the analyte is not bound to the binding partner and whichantibody, when bound to the binding partner, prevents the labelledmarker from binding to the binding partner.

The blocker may also be a nucleic acid (DNA or RNA) that binds to thebinding partner only when the analyte is not bound to the bindingpartner and which nucleic acid, when bound to the binding partner,prevents the labelled marker from binding to the binding partner.

Further, the blocker may also be a substance that modifies theunoccupied binding partner in such a fashion that it is no more able tobind the labelled marker, but which substance does not modify theoccupied binding partner in such a fashion that it would not be able toliberate the analyte bound thereto and subsequently bind the labelledmarker. The modifying substance may be a chemical compound or an enzyme.

A blocker that is a chemical compound may modify the binding partner byreacting with a specific residue in the binding partner. Such reactionsare for example alkylation of a free cysteine residue by a maleimidederivative or a iodoacetate derivative, nitration of a tyrosine residueby tetranitromethane, bromination of a tryptophan residue byN-bromosuccinimide or the iodination of a tryptophan residue by iodine.These examples of chemical modification of the binding partner are wellknown to the skilled art worker, and are widely reviewed in theliterature of the art. A general but not exhaustive description of thesetechniques is presented in reference [7]. Further, the chemical compoundmay be a reactive derivative of the analyte such as an aryl derivative[7], which binds to the binding partner first by biospecificrecognition, and later forms a covalent bond by virtue of the reactivegroup. If the reactive group is an arylazide, the formation of thecovalent bond can be controlled by photoactivation with ultraviolet orvisible light [8].

A blocker that is an enzyme may modify the binding partner for exampleby digestion, by adding a phosphate, by removing a phosphate, byglycosylation or by deglycosylation. Enzymes that digest the bindingpartner are for example proteases, such as trypsin, pepsin, papain,factor Xa, V8 or enterokinase. Further, the enzymes that digest thebinding partner may also be nucleases, if the binding partner or a partthereof is a nucleic acid (DNA or RNA). Such nucleases include forexample type II restriction endonucleases, exonuclease III, DNAse I, andRNAses. Enzymes that add a phosphate are for example kinases such as atyrosin kinase or a serine kinase. Enzymes that remove a phosphate arefor example phosphatases such as LAR protein tyrosine phosphatase.Enzymes that add or remove a glycosyl group are for example glycosylasesor glycosidases.

If the blocker is a molecule that binds non-covalently to the bindingpartner, its rate of dissociation from the binding partner must be lowerthan that of the analyte (at least five times as low, preferably morethan one hundred times as low, most preferably more that one thousandtimes as low).

If the blocker is a chemical compound or an enzyme, it must be able tomodify most of the unoccupied binding partners (at least more than 90%,preferably more than 95%, typically more than 99%, most preferably morethan 99.8%).

The term "inactivate" shall mean any of the above listed mechanismswhereby the blocker prevents the unoccupied binding partner from bindingthe labelled marker.

The "marker" is a molecule which is able to bind to the binding partneronly when the binding partner is not occupied by the analyte and when itis not inactivated by the blocker. Preferably, but not necessarily, themarker and the analyte bind mutually exclusively to the same bindingsite on the binding partner. The marker is either labelled before thereplacement reaction, or it can be labelled after the replacementreaction. The label can be for example a radio-isotope, an enzyme or afluorescent, phosphorescent or luminescent molecule.

Preferred Embodiments

The main steps of a preferred embodiment of the method according to theinvention are illustrated on Scheme 1.

The method according to this invention is particularly suitable for themeasurement of 17β-estradiol. In one such embodiment the analyte is17β-estradiol, the binding partner is for example a monoclonal antibodyspecific for 17β-estradiol, the blocker is for example17β-estradiol-6-carboxymethyl oxime, and the labelled marker is forexample Eu-labelled 17β-estradiol-6-carboxymethyl oxime.

Although a solid phase-bound antibody is used in the examples below, theinvention is not limited to this kind of embodiment. Equally well otherembodiments could be devised in which the incubation of the analyte withthe binding partner could take place in solution.

The invention is illuminated by the following examples.

EXAMPLE 1 Preparation of Europium-labelled 17β-Estradiol-6-carboxymethylOxime.

Europium chelate of4-[2-(4-aminophenyl)ethynyl]-2,6-bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridinewas a gift from H. Mikola, Wallac Oy. It was synthetized by the methodof Takalo et al. [9,10]. 6-oxoestradiol 6[O-(6-aminohexyl)oxime](abbreviated 6-AHO) was a gift from H. Mikola, Wallac Oy. It wasprepared according to the method of Mikola and Hanninen [11].Europium-labelled estradiol was a gift from H. Mikola, Wallac Oy. It wassynthetized from 6-AHO and the Europium chelate of4-[2-(4-aminophenyl)ethynyl]-2,6-bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridineusing water soluble carbodiimide as condensation agent in abuffer-dioxane solution and purified on a gel filtration column usingmethods of Mikola and Miettinen [12] and Mikola et al. [13].

EXAMPLE 2 Determination of Affinity Constants (Ka) of various MonoclonalAntibodies for Europium-labelled 17β-Estradiol-6-carboxymethyl Oxime.

Three commercially available monoclonal anti-17β-estradiol antibodieswere tested. These antibodies were all raised against position 6derivatives of 17β-Estradiol. The monoclonal antibodies used were069-A5406A (BiosPacific, California), 10-E15 (Fitzergald IndustriesInternational Inc., Massachusetts) and 2F9 (InterPharm LaboratoriesLtd., Israel) and they had been raised against the following immunogens:

    ______________________________________                                        Antibody     Immunogen (According to manufacturer)                            ______________________________________                                        069-A5406A   Estradiol-6-Bovine serum albumin                                   10-E15 Estradiol-6-Carboxymethyloxime-carrier                                 2F9 Estradiol-6-Carboxymethyloxime-                                            Bovine serum albumin                                                       ______________________________________                                    

The affinity constants of the monoclonal antibodies 069-A5406A, 10-E15and 2F9 for Europium-labelled 17β-Estradiol-6-carboxymethyl oxime (seeExample 1) were determined as follows:

All incubations were performed at 22° C. Antibodies were diluted inAssay buffer [50 mM Tris-HCl pH 7.75, 0.9% NaCl, 0.05% NaN₃, 0.01% Tween40, 0.05% Bovine gammaglobulin, 20 μM Diethylenetriaminepentaaceticacid, 0.5% Bovine serum albumin, 20 μg/ml Cherry Red] at a concentrationof 10 ng/ml. The diluted antibodies were placed in the wells of rabbitanti-Mouse IgG-coated microtitration strips (Wallac Oy, Turku, Finland),200 μl per well, and the strips were shaken in a plate shaker at 600rounds per minute for 2 hours. During the incubations, serial dilutionsof Europium-labelled estradiol were made. After the incubations, thestrips were washed four times with washing solution [0.9% (w/v) NaCl, 5mM Tris-HCl pH 7.75, 0.005% Tween]. 200 μl of each dilution of theEuropium-labelled estradiol were added to separate wells and the stripswere shaken in a plate shaker at 600 rounds per minute for 2 hours. Thestrips were washed four times with washing solution, after which 200 μlof Delfia Enhancement solution (Wallac) was added. After an incubationof 30 min the time-resolved fluorescence signals were read with a Platefluorometer (Wallac). The data were plotted as bound/free vs. boundEuropium-labelled estradiol and the affinity constants were calculatedfrom the slopes of the plots according to the method of Scatchard [14].

Results:

Affinity constants (Ka (L/mol)) for complexes between Eu-labelled17β-Estradiol-6-carboxymethyl oxime and the various monoclonalantibodies.

    ______________________________________                                               Mab     Ka                                                             ______________________________________                                               069-A5406A                                                                            6.7 × 10.sup.10                                            10-E15 2.5 × 10.sup.10                                                  2F9 9.4 × 10.sup.10                                                   ______________________________________                                    

EXAMPLE 3 Determination of Half-lives of Complexes Between VariousMonoclonal Antibodies and 17β-Estradiol, 17β-Estradiol-6- carboxymethylOxime or 17β-Estradiol-6-aminohexyl Oxime

The half-lives of complexes between monoclonal antibodies (069-A5406A,10-E15 or 2F9) and 17β-Estradiol (Sigma Chemical Company),17β-Estradiol-6-carboxymethyl oxime (abbreviated 6-CMO; Sigma ChemicalCompany) or 17β-Estradiol-6-aminohexyl oxime (abbreviated 6-AHO, seeExample 1) were determined as follows:

All incubations were performed at 22° C.; all dilutions were done inAssay buffer [50 mM Tris-HCl pH 7.75, 0.9% NaCl, 0.05% NaN₃, 0.01% Tween40, 0.05% Bovine gammaglobulin, 20 μM Diethylenetriaminepentaaceticacid, 0.5% Bovine serum albumin, 20 μg/ml Cherry Red]. Dilutedantibodies (10 ng/ml) were placed in the wells of rabbit anti-MouseIgG-coated microtitration strips (Wallac), 200 μl per well, and thestrips were shaken in a plate shaker at 600 rounds per minute for 2hours. The strips were washed four times with washing solution [0.9%(w/v) NaCl, 5 mM Tris-HCl pH 7.75, 0.005% Tween], after which 200 μl ofeither 100 nM 17β-estradiol, 10 nM 6-CMO or 10 nM 6-AHO were added tothe wells and the strips were shaken in a plate shaker at 600 rounds perminute for 60 min. The strips were washed four times with washingsolution, after which 200 μl of 10 nM Europium-labelled 17μ-Estradiolwas added. The strips were again shaken in the plate shaker at 600rounds per minute for varying times. The strips were taken from theplate shaker one at a time and washed four times with the washingsolution, after which Delfia Enhancement solution (200 μl per well;Wallac) was added. After the addition of the enhancement solution, eachstrip was incubated for 30 min before the time-resolved fluorescencesignals were read with a Plate fluorometer (Wallac). The data wereplotted as the natural logarithm of the signal (cps) vs. the elapsedtime in minutes (from the addition of Europium-labelled estradiol to thestart of the washes). Half-lifes were calculated from the slopes of theplots with the following formula:

Half-life (min)=[ln(2)]/-k , where k is the slope of the plot.

Results:

Half-lives (in minutes) of Mab-antigen complexes with the antiges17β-Estradiol, 6-CMO and 6-AHO.

    ______________________________________                                                Antigen                                                               Mab       17β-Estradiol                                                                           6-CMO   6-AHO                                        ______________________________________                                        069-A5406A                                                                              15             47      64                                             10-E15 9 127 93                                                               2F9 44 51 154                                                               ______________________________________                                    

For the purpose of this invention the Mab 10-E15 seemed most promisingbecause there is a 14-fold difference between the half-lives of itscomplexes with 17β-Estradiol (9 min) and 6-CMO (127 min).

EXAMPLE 4 Production of a Dose-response Curve for 17β-Estradiol

All incubations were performed at 22° C.; all dilutions were done inAssay buffer [50 mM Tris-HCl pH 7.75, 0.9% NaCl, 0.05% NaN₃, 0.01% Tween40, 0.05% Bovine gammaglobulin, 20 μM Diethylenetriaminepentaaceticacid, 0.5% Bovine serum albumin, 20 μg/ml Cherry Red]. Mab 10-E15(Fitzgerald Industries International Inc., Massachusetts) wasimmobilized in the wells of rabbit anti-Mouse IgG-coated microtitrationstrips (Wallac Oy, Turku, Finland), by adding 100 μl per well of a 100ng/ml dilution of the antibody, shaking in a plate shaker at 600 roundsper minute for 1 hour, and washing the strips four times with washingsolution [0.9% (w/v) NaCl, 5 mM Tris-HCl pH 7.75, 0.005% Tween]. 100 μlper well of 17β-Estradiol standards of the concentrations 0, 0.025,0.050, 0.075, 0.1, 0.2, 0.5 and 2 nM were added to the strips containingthe immobilized antibody, after which the strips were shaken in theplate shaker at 600 rounds per minute for 2 hours. The blockingreactions were initiated by adding 100 μl per well of a 10 μM solutionof 17β-estradiol-6-carboxymethyl oxime (Sigma Chemical Company). Thestrips were further shaken for 5 min and then washed with washingsolution four times. The replacement reaction was initiated by adding100 μl per well of 10 nM Europium-labelled 17β-estradiol, after whichthe strips were shaken for either 5 or 10 minutes and washed withwashing solution four times. 200 μl of Delfia Enhancement solution(Wallac) was added and the plates were shaken for 30 min after which thetime-resolved fluorescence signal was read with the Plate Fluorometer(Wallac).

Dose-response curves were prepared by plotting the signals against theconcentrations of the standards in an X-Y plot, see FIGS. 1A and 1B. Thefilled squares represent replacement time of 10 minutes and the opensquares represent replacement time of 5 minutes. FIG. 1A shows thecurves in the concentration range 0 to 0.5 nM and FIG. 1B shows thecurves over the whole measuring range of 17β-Estradiol standardconcentrations. Every point shown is an average of three paralleldeterminations. Error bars extend±1 SD of the mean value in each point.

The least detectable dose LDD was defined as the 17β-Estradiolconcentration which gives a signal that differs by two standarddeviations from the zero-concentration signal. This value is calculatedby dividing the standard deviation (SD) of the zero concentration signalby the slope of the linear portion of the standard curve.

It will be appreciated that the methods of the presented invention canbe incorporated in the form of a variety of embodiments, only a few ofwhich are disclosed herein. It will be apparent to the specialist thatother embodiments exist and do not depart from the spirit of theinvention. Thus, the described embodiments are only illustrative andshould not be construed as restrictive.

SCHEME 1 ##STR1##

What is claimed is:
 1. A method for the determination of analytescomprising the steps of a) contacting a sample containing said analytewith a binding partner specific for said analyte,b) adding a blocker,said blocker being able to inactivate the binding sites of the bindingpartner that are unoccupied, but not being able to inactivate thebinding sites of the binding partner that are occupied by the analyte,c) dissociating bound analyte from the binding partner, d) adding alabelled marker which is able to occupy the binding sites from which theanalyte was dissociated, but which is not able to occupy the bindingsites that were inactivated by the blocker, wherein step d can beperformed subsequently to or simultaneously with step c, and e)measuring a signal from the labelled marker bound to the bindingpartner, wherein said signal is directly proportional to an amount ofsaid analyte originally contained in said sample.
 2. The methodaccording to claim 1 wherein the binding partner is an antibody.
 3. Themethod according to claim 1 whe rein the binding partner is immobilizedto a solid phase.
 4. The method according to claim 1 wherein the blockeris bound to the binding partner by specific molecular recognition. 5.The method according to claim 4 wherein the blocker is non-covalentlybound to the binding partner, and the rate of dissociation of theblocker from the binding partner is at least five times lower than therate of dissociation of the analyte from the binding partner.
 6. Themethod according to claim 4 wherein the blocker is covalently bound tothe binding partner by virtue of a reactive group present in theblocker.
 7. The method according to claim 1 wherein the blocker is asubstance that chemically or enzymatically modifies the binding partnerso as to inactivate its binding site.
 8. The method according to claim 1wherein the analyte is a steroid.
 9. The method according to claim 8wherein the analyte is 17β-estradiol, the blocker is17β-estradiol-6-carboxymethyl oxime, and the labelled marker is labelled17β-estradiol-6-carboxymethyl oxime.